Decoding calcium signals by multifunctional CaM kinase.

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is one of the three major protein kinases coordinating cellular responses to hormones and neurotransmitters. It mediates the action of Ca2+ on neurotransmitter synthesis and release, on carbohydrate metabolism and on the cytoskeleton. CaM kinase has structural/functional properties that facilitate its response to distinctive attributes of Ca2+ signals which often involve transient increases that span a narrow concentration range and increases that are pulsatile rather than persistent. The kinase responds to the narrow working range of Ca2+ signals by the use of calmodulin as the Ca2+ sensor. It is activated by the binding of calmodulin to an autoinhibitory domain that keeps the kinase inactive in the basal state. The transient nature of the signal is accommodated by autophosphorylation of this autoinhibitory domain which allows the kinase to remain partially active after calmodulin dissociates and thereby switches it to a Ca(2+)-independent species. The pulsatile nature of Ca2+ signals may also be decoded by CaM kinase. Autophosphorylation traps calmodulin on autophosphorylated subunits by greatly reducing its off-rate. At high frequency of stimulation, calmodulin would remain trapped during the brief interval between Ca2+ oscillations and each successive rise in Ca2+ would recruit more calmodulin. This may enable a stimulus frequency dependent activation of CaM kinase.     

Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels

A dynamic positive feedback mechanism, known as 'facilitation', augments L-type calcium-ion currents (ICa) in response to increased intracellular Ca2+ concentrations. The Ca2+-binding protein calmodulin (CaM) has been implicated in facilitation, but the single-channel signature and the signalling events underlying Ca2+/CaM-dependent facilitation are unknown. Here we show that the Ca2+/CaM-dependent protein kinase II (CaMK) is necessary and possibly sufficient for ICa facilitation. CaMK induces a channel-gating mode that is characterized by frequent, long openings of L-type Ca2+ channels. We conclude that CaMK-mediated phosphorylation is an essential signalling event in triggering Ca2+/CaM-dependent ICa facilitation.     

High-affinity cholecystokinin type A receptor/cytosolic phospholipase A2 pathways mediate Ca2+ oscillations via a positive feedback regulation by calmodulin kinase in pancreatic acini.

In rat pancreatic acini, we previously demonstrated that depending on the agonist used, activation of cholecystokinin type A (CCKA) receptor (CCK-AR) results in the differential involvement of the cytosolic phospholipase A2 (cPLA2), phospholipase Cbeta1 (PLCbeta1) and Src/protein tyrosine kinase (PTK) pathways. The high-affinity CCK-AR appears to be coupled to the Gbeta/cPLA2/arachidonic acid (AA) cascade in mediating Ca2+ oscillations. The low-affinity CCK-AR is coupled to both the Galphaq/11/PLCbeta1/inositol 1,4,5-trisphosphate (IP3) to evoke intracellular Ca2+ release and the Src/PTK pathway to mediate extracellular Ca2+ influx. The objectives of this study were to provide evidence that cPLA2 is present in pancreatic acini and to evaluate the possibility that its activation results in Ca2+ oscillations and amylase secretion. Furthermore, we investigated the mechanism of Ca2+ oscillations mediated by the high-affinity CCK-AR. In rat pancreatic acini, immunoprecipitation studies using an anti-cPLA2 monoclonal antibody, demonstrated a cPLA2 band at the location of 110 kDa. A selective inhibitor of cPLA2, AACOCF3 (100 microM), inhibited production of AA metabolites, Ca2+ oscillations and amylase secretion elicited by the high-affinity CCK-AR agonist, CCK-OPE (10-1000 nM). In addition, through the repetitive release of intracellular Ca2+, CCK-OPE enhanced phosphotransferase activities of Ca2+/calmodulin-dependent protein kinase type IV (CaMK IV), which were inhibited by AACOCF3. The CaMK inhibitor, K252-a (1-3 microM), also abolished basal and CCK-OPE-stimulated CaMK IV activities. The CaM inhibitor, W-7 (100 microM), and K252-a inhibited Ca2+ oscillations and amylase secretion evoked by CCK-OPE without affecting the AA formation. Therefore, it appears that Ca2+ oscillations elicited by the high-affinity CCK-AR/Gbeta/cPLA2/AA pathway activate CaMK IV. Activated CaMK, in turn, regulates Ca2+ oscillations through a positive feedback mechanism to mediate pancreatic exocytosis.     

Novel Ca2+/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca2+/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca2+/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.     

Roles of Calmodulin and Calcium/Calmodulin-Dependent Protein Kinase in Flagellar Motility Regulation in the Coral Acropora Digitifera

In the corals Acropora spp., eggs secrete substances that induce sperm motility regulation. An elevation of intracellular pH ([pH]i) and a regulation of intracellular Ca²⁺ concentration ([Ca²⁺]) are involved in the sperm motility regulation cascade. However, the detailed molecular aspects of flagellar motility regulation have not been fully demonstrated in Acropora. In this study, we determined the presence and roles of both calmodulin (CaM) and calcium/calmodulin dependent-protein kinase (CaMK) in the sperm flagellar motility regulation of Acropora. A ⁴⁵Ca²⁺-overlay assay and an immunoblot analysis showed that sperm contain an acidic 16-kDa protein that was CaM, and an immunoblot analysis revealed the presence of CaMK in coral sperm. In addition, a specific inhibitor of CaMK, KN-93, and a CaM antagonist, W-7, inhibited sperm motility activation induced by NH₄Cl treatment. NH₄Cl treatment causes an increase in intracellular [pH]i of sperm, suggesting that CaM and CaMK are involved in sperm motility initiation caused by an increase in [pH]i. The involvement of CaM and CaMK in motility regulation in coral highlights the importance of these molecules throughout the animal kingdom.     

Glial cell line-derived neurotrophic factor increases intracellular calcium concentration. Role of calcium/calmodulin in the activation of the phosphatidylinositol 3-kinase pathway

Moderate increases of intracellular Ca2+ concentration ([Ca2+]i), induced by either the activation of tropomyosin receptor kinase (Trk) receptors for neurotrophins or by neuronal activity, regulate different intracellular pathways and neuronal survival. In the present report we demonstrate that glial cell line-derived neurotrophic factor (GDNF) treatment also induces [Ca2+]i elevation by mobilizing this cation from internal stores. The effects of [Ca2+]i increase after membrane depolarization are mainly mediated by calmodulin (CaM). However, the way in which CaM exerts its effects after tyrosine kinase receptor activation remains poorly characterized. It has been reported that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target protein kinase B (PKB) play a central role in cell survival induced by neurotrophic factors; in fact, GDNF promotes neuronal survival through the activation of the PI 3-kinase/PKB pathway. We show that CaM antagonists inhibit PI 3-kinase and PKB activation as well as motoneuron survival induced by GDNF. We also demonstrate that endogenous Ca2+/CaM associates with the 85-kDa regulatory subunit of PI 3-kinase (p85). We conclude that changes of [Ca2+]i, induced by GDNF, promote neuronal survival through a mechanism that involves a direct regulation of PI 3-kinase activation by CaM thus suggesting a central role for Ca2+ and CaM in the signaling cascade for neuronal survival mediated by neurotrophic factors.     

Ca2+/钙调蛋白依赖性蛋白激酶在细胞增殖中的作用

钙调蛋白(calmodulin,CaM)是Ca2 的受体蛋白,活化的CaM经Ca2 /CaM依赖性蛋白激酶(Ca2 /calmodulin dependent protein kinases,CaMKs途)径,影响细胞的生长和分裂。CaMKs在调节不同组织正常细胞及恶性细胞的细胞周期进程、核转录及信号转导的过程中发挥重要作用,通过不同机制及Ca2 /CaM依赖性激酶激酶诱导的相关级联反应影响多种细胞的增殖。对CaMKs主要成员CaMKI、CaMKII、CaMKIII、CaMKIV的生物学特点以及其在细胞增殖中作用的最新研究进展进行了综述。